PDGF was first recognized as a component of platelet .alpha. granules, which had growth promoting activity for smooth muscle cells and fibroblasts (Heldin and Westermark, Cell Regul 1: 555-566 (7-90)). It has also been implicated in the stimulation of connective tissue-derived cells in vitro (Ostman et al., J. Biol. Chem. 263(31): 16202-16208 (11-88)), as the major mitogenic protein for mesenchymal cells (Murray et al., U.S. Pat. Nos. 4,889,919 and 4,845,075), and as an inducer of cell multiplication and DNA synthesis in cultured muscle cells, fibroblasts and glial cells (Kelly et al, PCT Application W090/14425 (Nov. 29, 1990)). It has also been shown to be involved in the wound healing response (Ross et al., N. Eng. J. Med. 295: 369 (1976)), and may be involved in a causative role for the development of proliferative lesions of artherosclerosis (Ross) supra. Others have suggested that this molecule may be a mediator of tumor development as well as in nonmalignant proliferative disorders (Heldin et al., supra).
The PDGF molecule has been very well characterized. It is known to exist as a heterodimer of an "A" chain and a "B" chain, connected to each other via disulphide bonds. The dimer, sometimes referred to as "PDGF-AB", has a molecular mass of about 30 KDa. Amino acid sequences are known for both the A and B chains, as shown, e.g., by Murray et al., U.S. Pat. Nos. 4,889,919 and 4,845,075, the disclosures of which are incorporated by reference. The mature chains contain slightly more than 100 amino acids, and are about 60% homologous. Heldin et al., supra.
Dimers PDGF-AA and PDGF-BB have been produced via recombinant means, and have also been isolated from natural sources (see Murray et al., supra; Heldin et al., supra). The various dimers, or "isoforms" differ in functional properties and secretory behavior.
The mechanism by which PDGF acts on cells has received intensive scrutiny, and it has been established that there are two receptors for PDGF, the ".alpha." and ".beta." receptors. The .alpha. receptor binds all isoforms, whereas .beta. receptor does not bind PDGF-AA, binds PDGF-AB with low affinity, and PDGF-BB with high affinity (Heldin et al., supra; Ostman et al., supra). The receptor is synthesized as a 140 KDa precursor protein which matures to one of 170 KDa, and the .beta. receptor is recognized as a precursor of 160 KDa, and a mature molecule of 180 KDa. cDNA for both receptors has also been isolated (Heldin et al., supra; Kelly et al., supra).
The structure of the receptors is linked to their function; both comprise five immunoglobulin like domains (extracellular portion), and intracellular portions containing protein tyrosine kinase domains with characteristic insert sequences which have no homology to kinase domains (Yarden et al., Nature 323: 226-232 (1986); Matsui et al., Science 243: 800-803 (1989); Claesson-Welsh et al., PNAS 86: 4917-4921 (1989). When PDGF binds to these receptors, dimerization of the receptor molecules is induced, followed by kinase activation and autophosphorylation of the receptors (Heldin et al., J. Biol. Chem. 264: 8905-8912 (1989); Seifert et al., J. Biol. Chem. 264: 8771-8778 (1989); Bishayee et al., J. Biol. Chem. 264: 11699-11705 (1989)).
The many mechanisms with which PDGF is involved and the manner in which it reacts with its receptors suggests further study as to how this interaction can be modified One approach to this type of study involves the use of agonists and antagonists. The molecules, using the definitions employed by Kelly et al., supra, either mimic the effect of PDGF (agonists), or block the interaction of receptor and ligand (antagonists).
The art has long recognized that agonists and antagonists for various materials exist, and Kelly et al., via their discussion, de facto assumes that these exist for PDGF. Review of the literature indicates, however, that no proteinaceous agonists and antagonists to PDGF are taught. For the reasons described supra, it would be desirable to have such material available.
The two patents to Murray et all, cited supra discuss potential amino acid substitution of cysteine residues in the monomeric chains, provided that these substitutions do not destroy the biological activity of the molecules. The '919 patent generally teaches modifications of PDGF AA molecules. Neither reference teaches that modified dimers of PDGF have antagonistic activity against wild type PDGF.
It has now been found that portions of the PDGF chains, i.e., peptides derived therefrom, can be used as both agonists and antagonists. It has also been found that manipulation of the dimeric structure of the PDGF molecule leads to the creation of monomeric molecules which are PDGF agonists. Additionally, it has been found that in dimeric PDGF molecules, there is a "cross" bond of the second cysteine of each monomer with the fourth cysteine of the other monomer, and that elimination of one of these bond creates a dimeric molecule which competes with wild type PDGF. PDGF-B agonists comprising amino acids 97-180 of the PDGF-B monomer having the cysteines at amino acid residues 124 and 133 substituted, nucleic acid molecules encoding the agonists, plasmids, transformed host cells, and methods for causing receptor dimerization and autophosphorylation in a cell having PDGF-.beta. receptors on its surface comprising administering the PDGF-B agonist form part of the invention. Also described are new PDGF heterodimers, methods and systems for producing these. These are the key features of the invention described herein, which will be seen from the disclosure which follows.